Defibrotide for use in prophylaxis and/or treatment of graft versus host disease (gvhd)

ABSTRACT

Defibrotide for use in prophylaxis and/or treatment of Graft versus Host Disease (GVHD) in humans is disclosed, preferably in hematopoietic stem cell transplantation (HSCT), more preferably allogeneic hematopoietic stem cell transplantation. Graft versus Host Disease of the invention (GVHD) can be acute aGVHD and/or chronic cGVHD, preferably acute.

The present invention relates to defibrotide for use in prophylaxisand/or treatment of Graft versus Host Disease (GVHD) in humans,preferably in hematopoietic stem cell transplantation (HSCT), morepreferably allogeneic hematopoietic stem cell transplantation. Graftversus Host Disease of the invention (GVHD) can be acute aGVHD and/orchronic cGVHD, preferably acute.

STATE OF THE ART

Graft versus Host Disease (GVHD) is the most frequent complication afterallogeneic haematopoietic stem cell transplantation (HSCT). GVHD canoccur despite aggressive immunosuppressive prophylaxis even when thedonor is a perfectly matched (HLA identical) sibling. It is aconsequence of interactions between antigen presenting cell of recipientand mature-cell of donor.

Traditionally, GvHD is regarded as an epithelial cell disease. The threeorgans mainly involved in acute GvHD are skin, gastrointestinal tractand liver. All of them share the common feature of presenting a barrierto the ‘environment’ of the host. Chronic GvHD similarly attacks barriertissues. In the chronic sicca syndrome of the eye, the oral, intestinalor genital mucosal compartments, as well as in bronchiolitis obliterans,the epithelial barrier of the mucosa is involved. Furthermore, GvHD isoften considered as a single disease, split into two phases: an acutephase of GvHD occurring early after HSCT, and a chronic phase in whichGvHD appears later in the course of transplantation.

Finally, in the traditional view, GvHD is cytokine-triggered. Damageinduced by the conditioning preparative regimen used beforetransplantation and infections causes the release of diverse cytokineswhich are responsible for an inflammatory process, enhancing the GvHDreaction.

Defibrotide, a polydisperse mixture of single-stranded oligonucleotides,has a protective effect on activated endothelial cells. Preclinicalstudies revealed that defibrotide protects these cells againstchemotherapy-induced cell death and activation, and downregulates thegene expression, protein level and activity of endothelial cell-triggerslike heparanase. Defibrotide reduces procoagulant activity and increasesfibrinolytic properties of stimulated endothelial cells withoutexacerbating systemic bleeding. Clinical studies of defibrotide inpatients with severe hepatic Veno-Occlusive Disease (VOD) following HSCThave reported complete resolution in 36 to 50% of patients, and Day+100survival in 35 to 42%. The prophylactic role of defibrotide has not beenstudied prospectively, but appears promising. We evaluated the efficacyand safety of defibrotide in preventing VOD against standard practice(no VOD prophylaxis) in a high-risk pediatric HSCT population.

DEFINITION

The term defibrotide identifies a polydeoxyribonucleotide that isobtained by extraction from animal and/or vegetable tissues but whichmay also be produced synthetically; the polydeoxyribonucleotide isnormally used in the form of an alkali-metal salt, generally a sodiumsalt, and generally has a molecular weight of 13 to 30 kDa (CAS RegistryNumber: 83712-60-1). Preferably, defibrotide is obtained according toU.S. Pat. No. 4,985,552 and U.S. Pat. No. 5,223,609 and/or presents thephysical/chemical characteristics described in the same U.S. Pat. No.4,985,552 and U.S. Pat. No. 5,223,609, herein incorporated by reference.More in particular, defibrotide is a mixture of polydeoxyribonucleotideshaving formula of random sequence:

P1-5, (dAP)₁₂₋₂₄, (dGP)₁₀₋₂₀, (dPp)₁₃₋₂₆, (dCP)₁₀₋₂₀

whereinP=phosphoric radical,dAp=deoxyadenylic monomer,dGp=deoxyguanylic monomer,dTp=deoxythymidinic monomer,dCp=deoxycytidynic monomer.and/or shows the following chemical/physical characteristics:

Electrophoresis=homogeneous anodic mobility, and/or extinctioncoefficient, E₁ cm^(1%) at 260±1 nm nm=220±10, and/orE₂₃₀/E₂₆₀=0.45±0.04, and/or coefficient of molar extinction (referred tophosphorous) ε(P)=7.750±500, and/or rotatory power [α]_(D) ^(20°)=53°±6;and/or reversible hyperchromicity, indicated as % in native DNA and/orh=15±5.

The term humans identifies any subject as adult subjects and pediatricpopulation, wherein with the term pediatric population is intended thepart of population from birth to eighteen (18) years old.

The term aGVHD identifies acute Graft versus Host Disease;

The term cGVHD identifies chronic Graft versus Host Disease; and

The term allogenic referred to hematopoietic stem cell transplantation(HSCT) identifies transplant from an healthy subject to a (patient)recipient.

DESCRIPTION OF THE INVENTION

The present invention refers to defibrotide for use in prophylaxisand/or treatment of Graft versus Host Disease (GVHD) in humans,preferably in hematopoietic stem cell transplantation (HSCT). Graftversus Host Disease of the invention (GVHD) can be acute aGVHD and/orchronic cGVHD, preferably acute.

According to the invention, defibrotide can be administered to humansbefore and/or after hematopoietic stem cell transplantation (HSCT).

We have recently performed an international, randomized, controlled,open-label trial, wherein we compared defibrotide (Gentium S.p.A.)prophylaxis with no prophylaxis in pediatric HSCT patients at high riskfor developing VOD. The primary endpoint was the incidence of VOD byDay+30 post-HSCT, adjudicated by a blinded, independent reviewcommittee. Secondary endpoints included graft-versus-host disease(GVHD), VOD-related organ failure and mortality.

A total of 356 patients met the inclusion criteria and gave informedconsent to be randomized to the i.v. defibrotide arm (n=180) or thecontrol arm (n=176). VOD was reported in 22 patients (12%) in thedefibrotide arm and in 35 patients (20%) in the control arm (competingrisk, P=0.05; Kaplan-Meier, P=0.05). The incidence and severity of acuteGVHD were significantly reduced (P=0.005 and P=0.003, respectively) inthe allogeneic recipients. VOD-associated organ failures were lower inthe defibrotide arm with a significant reduction in the incidence ofrenal failure (1% vs. 6%, P=0.02). A significantly higher Day+100mortality was observed in patients with VOD (25% vs. 6%; P<0.001).Although mortality after VOD diagnosis was lower in the defibrotide arm(4 vs. 10 patients, P=0.1), overall mortality was similar in the twoarms. There was no difference in the incidence of adverse events betweenarms (87% vs. 88%).

Defibrotide reduced the incidence of VOD by 40%, as well as theincidence and severity of acute GVHD, and has a good safety profile.(ClinicalTrials.gov number, NCT00272948.)

Methods Study Design

This study was a phase 3, multicenter, randomized trial, supportedGentium S.p.A. The study protocol and patient consent form were approvedby the research ethics committees of the participating centers andwritten informed consent was obtained from patients and/or their legalrepresentatives prior to study entry.

Treatment assignments were generated by a central data manager with acomputer algorithm accessing a randomization sequence. Randomization was1:1, stratified by center and the diagnosis of osteopetrosis. Anindependent data and safety monitoring board (DSMB) of three experthematologists and a statistician reviewed the safety and mortality dataat predetermined intervals. A blinded independent review committee (IRC)of three expert hematologists reviewed and adjudicated diagnosis for allsuspected and documented VOD cases. An independent statisticianperformed the data analysis.

Eligible patients were aged <18 years with myeloablative conditioningfor allogeneic or autologous HSCT, who had at least one risk factor forVOD: pre-existing liver disease, second myeloablative HSCT, allogeneicHSCT for leukemia beyond the second relapse, conditioning with busulfanand melphalan, previous treatment with GO, and diagnoses of inheritedMAS, adrenoleukodystrophy, or osteopetrosis.

Treatment

For patients randomized to the open-label defibrotide arm, the studydrug was administered daily at 25 mg per kilogram of body weight per dayin four divided intravenous infusions. Defibrotide prophylaxis startedon the same day as the pre-transplant conditioning regimen and wascontinued until Day+30 or, if discharged from hospital before Day+30,for a minimum of 14 days. Patients in the control arm received noprophylaxis against VOD.

Treatment with systemic t-PA, therapeutic-dose heparin or otherantithrombotics was not permitted. Ursodiol was permitted.

Patients in either arm who developed VOD received treatment withdefibrotide at 25 mg per kilogram per day until complete resolution ofall symptoms or death.

Outcome Measures

The primary endpoint was the incidence of VOD by Day+30. VOD wasassessed by the Investigator according to modified Seattle criteria,defined as the presence of two or more criteria: bilirubin >2 mg perdeciliter (>34 pmol per liter), hepatomegaly, ascites, and/orunexplained weight gain >5% from baseline (modified from >2% weight gainin the original Seattle criteria²).

Laboratory parameters, including bilirubin, were measured at baseline(before conditioning) and at least weekly post transplant. All patientsunderwent abdominal ultrasonography at baseline and after transplant,when VOD was clinically suspected, to confirm presence of hepatomegalyand ascites. The blinded ultrasound reports and clinical data were sentto the IRC for adjudication of suspected and diagnosed VOD cases. TheIRC assessments were used in the primary efficacy analysis.

The secondary and exploratory efficacy parameters included:

(a) The incidence and severity of GVHD (International Bone MarrowTransplant Registry index) by Day+100 and Day+180.

(b) MOF and mortality by Day+100; although this study was not powered toassess MOF or mortality, these endpoints were analyzed together using acomposite scoring system for the assessment of VOD severity. This systemassigned one point for each organ failure in patients with VOD:respiratory failure (oxygen requirement and/or ventilator dependence),renal failure (the doubling of baseline creatinine level and/or dialysisdependence) and encephalopathy; five points were assigned for death(regardless of cause of death). Patients without VOD were assigned zero.Each composite score component was also analyzed individually.

(c) The incidence of transplant-associated microangiopathy (TAM)³¹ byDay+180.

Adverse events were recorded until Day+180 and coded using MedDRA terms.

Statistical Analysis

Analyses were performed as specified in the protocol and statisticalanalysis plan. The sample size was estimated based on the primaryendpoint (VOD incidence by Day+30). VOD rates were estimated at 30% inthe control arm and 15% in the defibrotide arm. Assuming a one-sidedlevel of significance at 0.025, power of 80%, and a 10% dropout rate,135 patients per arm were required. Because the incidence of VOD inchildren was unclear when the study was designed, the protocolincorporated a planned adaptive interim analysis, following thealgorithm of Denne, to be reviewed by an independent DSMB when 120patients per arm reached the Day+30 primary endpoint. The DSMBrecommended to increase the sample size to a total of 360 randomizedpatients.

All data analyses were performed on the intent-to-treat (ITT)population, which included all patients with informed consent randomizedinto the study. Comparison against the defibrotide arm for the primaryefficacy analysis was performed using a cumulative competing riskapproach by Kalbfleisch and Prentice as discussed in Tai et al., wheredeath not due to VOD, discontinuing the study due to an adverse event,and receipt of second transplant due to transplant failure, wereconsidered competing risks. A supportive analysis was also performedusing the log-rank test with Kaplan-Meier estimates of VOD rate, wherepatients not experiencing VOD were censored at Day+30, last knownfollow-up, or at the time of competing risk, whichever was earlier. Asecondary per protocol (PP) analysis was also planned, excludingprematurely withdrawn ITT patients and ITT patients with seriousprotocol violations.

The secondary efficacy analyses on the incidence and severity of GVHDwere analyzed using the Z-test for proportions and the two-sampleWilcoxon rank sum test, respectively.

Patients with no GVHD were assigned a severity value of 0.

The analysis on the VOD composite score was performed using an exactWilcoxon's test with a two-sided 5% level of significance.

Each composite score component was analyzed using the chi-square test.Kaplan-Meier distributions of time to death by Day+100 for each studyarm were compared using the log-rank test, where surviving patients werecensored at Day+100 or last known follow-up, whichever was earlier.

The safety population was defined for the defibrotide arm as allrecipients of at least one dose of study drug, and for the control armas all assigned patients. Safety parameters were assessed usingfrequency tables for serious and non-serious adverse events (AEs),laboratory tests, and survival up to Day+180, where survival is definedas the time in days between HSCT and death from any cause.

Results Patient Characteristics

Between January 2006 and January 2009, 360 patients were randomized tothe study in 28 centers in 11 countries in Europe. In the defibrotidearm, 181 patients were randomized, one in error (without consent),resulting in 180 patients in the ITT population. In the control arm, 179patients were randomized, three in error (without consent), resulting in176 patients in the ITT population.

The mean age of the study population was 6.6 years. Patients were evenlydistributed with respect to age and gender across the study arms (Table1). The primary diseases, type of donor, type of graft, and incidence ofrisk factors for VOD were similar between arms.

Patients randomized to the defibrotide arm received study drug for amean duration of 32.4 days (median, 35.0). When defibrotide was used fortreatment of VOD, the mean duration of therapy was 28.7 days (median,23.0) for the defibrotide arm and 21.7 days (median, 18.5) for thecontrol arm. The number of patients using concomitant ursodiol wassimilar between arms.

VOD Incidence:

The incidence of VOD was reduced by 40% in the defibrotide arm comparedwith the control arm. Analysis of the ITT population showed asignificantly lower incidence of VOD by Day+30 in the defibrotide armversus the control arm (cumulative incidence, 12% vs. 20%, respectively,according to modified Seattle criteria; competing risk, P=0.05;Kaplan-Meier, P=0.05; Table 2). Analysis of the PP population alsoshowed statistically significant results (competing risk, P=0.02; KaplanMeier, P=0.02).

To explore consistency of results across different criteria, anadditional subset analysis was performed applying Baltimore criteria,where bilirubin >2 mg per deciliter (>34 pmol per liter) is required forVOD diagnosis, with two of other three criteria, hepatomegaly, ascitesor weight gain >5%. We identified fewer VOD cases with Baltimorecriteria (7% in the defibrotide arm, 13% in the control arm; P=0.09;Table 2). While not significant, these data are consistent with the 40%reduction in VOD observed with modified Seattle criteria.

Effect of Defibrotide on GVHD The patients who received defibrotideprophylaxis experienced a significantly lower incidence and severity ofaGVHD by Day+100. In allogeneic HSCT recipients from the ITT population,the incidence and severity (Grades 1 to 4) of aGVHD were significantlylower in the defibrotide arm than in the control arm (P=0.005 andP=0.003, respectively), even when Grade 1 aGVHD was excluded (Table 3).There was no difference in the incidence of chronic GVHD (cGVHD) byDay+180 between study arms.

Despite its protective effects against aGVHD, defibrotide did notinterfere with the desirable graft versus leukemia effect. The combinedDay+100 relapse rates of acute lymphoblastic leukemia, acute myeloidleukemia, other leukemias and myelodysplastic syndromes were 6% for thedefibrotide arm versus 8% for the control arm, and combined Day+180relapse rates were 7% and 10%, respectively.

VOD-Associated MOF and Death:

To assess the effects of defibrotide on the severity of VOD, apre-specified composite scoring system was used to quantifyVOD-associated MOF and death up to Day+100. Overall, the defibrotide armhad fewer VOD-associated organ failures and deaths, resulting in astatistically significant reduction of scores (Wilcoxon test, P=0.03) inthe ITT population (Table 4). Specifically, the defibrotide arm showed astatistically significant lower incidence of renal failure compared withthe control group (1% vs. 6%; P=0.02). Severe VOD-associated MOF byDay+100 was significantly higher in patients who fulfilled Baltimorecriteria (60% vs. 32%; chi-square test, P=0.04).

VOD-associated mortality at Day+100 was lower in the defibrotide armthan the control arm (2% and 6%, respectively), but these results werenot statistically significant (Table 4). Overall mortality was similarin the two arms when assessed at Day+100 and Day+180

-   -   Mortality was significantly higher in patients with VOD at        Day+100 than in those without VOD (25% vs. 6%, respectively;        P<0.0001).

Overall, most fatal AEs were in the categories “Neoplasms, Malignant andUnspecified” (7% and 8% in the defibrotide and control arms,respectively), and “Infections and Infestations” (3% and 6%,respectively).

TAM

There was no difference in the incidences of TAM (3% and 4% in thedefibrotide and control arms, respectively) by Day+180.

Safety

The safety population included 177 patients of the defibrotide arm andall 176 patients in the control arm. The incidences of AEs (Table 5),and of serious adverse events (SAEs) and events leading to studydiscontinuation were similar between arms. A total of 207 SAEs werereported in 108 patients assigned to the defibrotide arm and 231 werereported in 103 control patients. Although the most common AE consideredby the Investigator to be possibly, likely or certainly related todefibrotide was hemorrhage, the incidence of hemorrhage was similarbetween arms.

Discussion

Although HSCT offers the only available cure for many diseases, theprocedure remains limited by regimen-related toxicities, including GVDand hepatic VOD. Indeed, our results show a nearly four-fold highermortality rate in patients with VOD compared with patients without VOD,and confirm previous observations.

In this study, prophylactic defibrotide reduced the incidence andseverity of aGVHD. Damage to the endothelial cells of skin,gastrointestinal tract and liver from conditioning regimens contributesto the onset of aGVHD. Defibrotide's anti-inflammatory and protectiveeffect on endothelial cells and down regulation of heparanase geneexpression could explain these results, which appear highly consistentwith the proposed mechanism of action of defibrotide.

Overall, defibrotide was well tolerated. Hemorrhages occurred with asimilar frequency in both study arms, strongly suggesting that theseevents are more likely related to SCT and VOD than to the administrationof defibrotide.

Object of the present invention is therefore defibrotide for use inprophylaxis and/or treatment of Graft versus Host Disease (GVHD) inhumans, preferably in hematopoietic stem cell transplantation (HSCT),more preferably allogeneic hematopoietic stem cell transplantation.Graft versus Host Disease of the invention (GVHD) can be acute aGVHDand/or chronic cGVHD, preferably acute.

Defibrotide according to the invention can be administered before and/orafter hematopoietic stem cell transplantation (HSCT), preferably beforeand/or after allogeneic hematopoietic stem cell transplantation.

Defibrotide according to the invention is preferably administered in adose ranging from 10 to 60 mg/kg per day, preferably in a dose rangingfrom 20 to 40 mg/kg per day and more preferably in a dose of about 25mg/kg per day; the kg refers to the body weight of the patient.According to the invention, defibrotide can be administered in a singleor repeated dose/s per day, preferably in four (4) doses per day.

According to the invention defibrotide is preferably administeredintravenously, more preferably is formulated in aqueous solution form.

REFERENCES

-   1. Copelan E A. Hematopoietic stem-cell transplantation. N Engl J    Med 2006;354(17):1813-26.-   2. James L. M. Ferrara, M. D.a,*, John E. Levine, M. D.b, Pavan    Reddy, M. D.c, and Ernst Holler, M. D.d Graft-versus-Host Disease.    Lancet. 2009 May 2; 373(9674): 1550-1561-   3. Stephanie J. Lee and Mary E. D. Flowers. Recognizing and Managing    Chronic Graft-Versus-Host Disease Hematology 2008: 134-41-   4. McDonald G B, Sharma P, Matthews D E, Shulman H M, Thomas E D.    Venocclusive disease of the liver after bone marrow transplantation:    diagnosis, incidence, and predisposing factors. Hepatology    1984;4(1):116-22.-   5. McDonald G B, Hinds M S, Fisher L D, et al. Veno-occlusive    disease of the liver and multiorgan failure after bone marrow    transplantation: a cohort study of 355 patients. Ann Intern Med    1993;118(4):255-67.-   6. Coppell J A, Richardson P G, Soiffer R, et al. Hepatic    veno-occlusive disease following stem cell transplantation:    incidence, clinical course, and outcome. Biol Blood Marrow    Transplant 2010; 16(2): 157-68.-   7. Eissner G, Multhoff G, Gerbitz A, et al. Fludarabine induces    apoptosis, activation, and allogenicity in human endothelial and    epithelial cells: protective effect of defibrotide. Blood    2002;100(1):334-40.-   8. Mitsiades C S, Rouleau C, Echart C, et al. Preclinical studies in    support of defibrotide for the treatment of multiple myeloma and    other neoplasias. Clin Cancer Res 200915(4)1210-21.-   9. Ostrovsky O, Shimoni A, Rand A, Vlodaysky I, Nagler A. Genetic    variations in the heparanase gene (HPSE) associate with increased    risk of GVHD following allogeneic stem cell transplantation: effect    of discrepancy between recipients and donors. Blood    2010;115(11):2319-28.-   21. Echart C, Graziadio B, Somaini S, et al. The fibrinolytic    mechanism of defibrotide: effect of defibrotide on plasmin activity.    Blood Coagul Fibrinolysis 2009;20(8):627-34.-   22. Falanga A, Vignoli A, Marchetti M, Barbui T. Defibrotide reduces    procoagulant activity and increases fibrinolytic properties of    endothelial cells. Leukemia 2003;17(8):1636-42.-   30. Rowlings P A, Przepiorka D, Klein J P, et al. IBMTR severity    index for grading acute graft-versus-host disease: retrospective    comparison with Glucksberg grade Br J Haematol 1997 97(4):855 - 64.-   31. Ruutu T, Barosi G, Benjamin R, et al. Diagnostic criteria for    hematopoietic stem cell transplant-associated microangiopathy:    results of a consensus process by an International Working Group.    Haematologica 2007;Jan(92(1)):95-100.-   35. Denne J S. Sample size recalculation using conditional power.    Stat Med 2001;20(17-18):2645-60.-   36. Tai B C, Machin D, White I, Gebski V. Competing risks analysis    of patients with osteosarcoma: a comparison of four different    approaches. Stat Med 2001;20(5):661-84.-   39. Ertault-Daneshpouy M, Leboeuf C, Lemann M, et al. Pericapillary    hemorrhage as criterion of severe human digestive graft-versus-host    disease. Blood 2004;103(12):4681-4.

TABLE 1 Baseline Demographics of the Study Participants. ControlDefibrotide Arm Arm Variable (N = 180) (N = 176) Age - yr Mean (SD) 6.5(5.2) 6.7 (5.4) Median 5.1 4.6 Range <1-18 <1-18 Age category - No.Infant and toddlers (28 days to 2 46 (26) 41 (23) (%) years) Children(>2 and ≦11 years) 91 (51) 95 (54) Adolescents (>11 years) 43 (24) 40(23) Female gender - Female 70 (39) 75 (43) No. (%) Primary disease -Neuroblastoma 34 (19) 33 (19) No. (%) Acute myelogenous leukemia 31 (17)42 (24) Acute lymphoblastic leukemia 26 (14) 22 (13) Other leukemia 8(4) 5 (3) Myelodysplastic syndrome 20 (11) 11 (6) Familialhemophagocytic 6 (3) 12 (7) lymphohistiocytosis Other inherited MAS 4(2) 3 (2) Soft tissue sarcoma 9 (5) 8 (5) Osteopetrosis* 7 (4) 6 (3)Adrenoleukodystrophy 1 (1) 1 (1) Other 34 (19) 33 (19) Type of donor -Matched related donor 35 (19) 25 (14) No. (%)† Matched unrelated donor55 (31) 61 (35) Mis-matched related donor 14 (8)  10 (6)  Mis-matchedunrelated donor 18 (10) 21 (12) Autologous HSCT 53 (29) 55 (31) Type ofgraft - No. Bone marrow 79 (44) 81 (46) (%)† Peripheral blood stem cells74 (41) 77 (44) Umbilical cord blood 16 (9)  10 (6)  T-cell depletedstem cells 6 (3) 4 (2) Immunosuppression - ATG-Horse 2 (1) 2 (1) No. (%)ATG-Rabbit 65 (36) 80 (45) Campath (alemtuzumab) 9 (5) 5 (3)Cyclosporine A 100 (56)  104 (59)  Methotrexate 56 (31) 65 (37) OKT-3 11(6)  7 (4) Other‡ 31 (17) 28 (16) Conditioning agent - Busulfan IV 80(44) 81 (46) No. (%) Busulfan PO 46 (26) 44 (25) Melphalan 126 (70)  114(65)  Cyclophosphamide 84 (47) 80 (45) Etoposide 22 (12) 25 (14) TotalBody Irradiation 17 (9)  18 (10) Fludarabine 34 (19) 40 (23) Treosulfan13 (7)  13 (7)  Other§ 34 (19) 31 (18) VOD high risk Secondmyeloablative transplant 25 (14) 23 (13) criteria - No. (%) AllogeneicHSCT for leukemia 17 (9)  11 (6)  Pre-existing liver disease 41 (23) 54(31) Prior abdominal irradiation 9 (5) 8 (5) Prior treatment withgemtuzumab 11 (6)  5 (3) Conditioning with busulfan and 106 (59)  99(56) melphalan Osteopetrosis 7 (4) 6 (3) Inherited MAS 10 (6)  15 (9) Adrenoleukodystrophy 1 (1) 1 (1) ATG denotes antithymocyte globulin,HSCT hematopoietic stem cell transplantation, MAS macrophage activationsyndromes. *Osteopetrosis was a stratification factor. †Five patients(3%) in the defibrotide arm and 4 patients (2%) in the control arm werewithout transplant data (did not proceed to myeloablative transplant orwithdrew conditioning before transplant). ‡Other immunosuppressants:mycophenolate mofetil, tacrolimus sirolimus, antilymphocyte globulin,prednisone/steroids, m-PDN and rituximab. §Other conditioning agents:thiotepa, alemtuzumab, radioimmunoantibodym carboplatin, rituximab, ATG,clofarabine, Endoxan, dexamethasone, amsacrine, aracytabine, muromonab Cand BCNU.

TABLE 2 Incidence of Veno-Occlusive Disease (VOD) Up To Day + 30 PostHematopoietic Stem-Cell Transplantation (HSCT). Defibrotide VODDiagnosis Arm Control Arm P value VOD by Day+30/ VOD diagnosed - No./N22/180 (12) 35/176 (20) IRC (ITT)* (%) Competing risk - % (95% 13 (8,19) 20 (15, 27) 0.05 CI)† Kaplan-Meier - % (95% 13 (9, 19) 20 (15, 27)0.05‡ CI) VOD by Day+30/ VOD diagnosed - No./N 18/159 (11) 34/166 (20)IRC (PP)§ (%) Competing risk - % (95% 11 (7, 17) 20 (15, 28) 0.02 CI)†Kaplan-Meier - % (95% 11 (7, 18) 21 (15, 28) 0.02‡ CI) VOD in patients1/7 (14) 4/6 (67) with osteopetrosis- No./N (%)¶ VOD by type ofAllogeneic HSCT 15 (8) 25 (14) donor - No. (%)* Autologous HSCT 7 (4) 10(6) VOD diagnosis Weight gain >5% 19 (11) 30 (17) criteria met - Ascites17 (9) 26 (15) No. (%)* Hepatomegaly 19 (11) 30 (17) Increased bilirubin13 (7) 22 (13) 0.09 VOD by Age Infants 9/46 (20) 11/41 (27) distributionChildren 10/91 (11) 16/95 (17) No./N (%) Adolescents 3/43 (7) 8/40 (20)*Intent to treat (ITT) population, N = 180 defibrotide arm and N = 176control arm. †Confidence interval (CI) by in transformation. ‡P value oflog-rank test (from Kaplan-Meier estimator). §Per protocol (PP)population, N = 159 defibrotide arm and N = 166 control arm. ¶N denotesnumber of patients with osteopetrosis; N = 7 defibrotide arm and N = 6control arm.

TABLE 3 The Incidence of Acute and Chronic Graft-Versus-Host disease(GVHD) in Allogeneic Hematopoietic Stem-Cell Transplantation (HSCT)Patients.* Defibrotide Arm Control Arm (N = 122) (N = 117) GVHDclassification No. (%) No. (%) P value Acute GVHD by Day + 100 57 (47)76 (65) 0.005† Acute GVHD severity Grade 1 30 (25) 33 (28) Grade 2 18(15) 30 (26) Grade 3 5 (4) 9 (8) 0.003‡ Grade 4 4 (3) 4 (3) Grades 2 to4¶ 27 (22) 43 (37) 0.01† Chronic-GVHD by Day + 180 16 (13) 17 (15) 0.8§*In the ITT population of all patients (allogeneic and autologous HSCTpatients), acute GVHD was present in 32% (57/180) of patients in thedefibrotide arm and 43% (76/176) of patients in the control arm; P =0.03. †P value from chi-square test for incidence of GVHD by Day + 100.‡P value from Wilcoxon test for grading of GVHD by Day + 100. §P valuefrom chi-square test for incidence of GVHD by Day + 180. ¶In a subset ofgrade 2 to 4 acute GVHD (excluding grade 1 mild cases), incidence was P= 0.01 and severity was P = 0.02 between the two arms.

TABLE 4 Veno-Occlusive Disease (VOD)-Associated Multiple Organ Failureand Death Up To Day + 100.* Defibrotide Arm Control Arm (N = 180) (N =176) Event No. (%) No. (%) Respiratory failure 11 (6)  15 (9) Renalfailure 2 (1) 10 (6) Encephalopathy 1 (1)  3 (2) Mortality 4 (2) 10 (6)No organ failure or mortality 169 (94)  159 (90) *Composite score inpatients with VOD: 1 point for each organ failure; 5 points formortality. Wilcoxon test for VOD-associated multiple organ failure anddeath in all patients at Day + 100: defibrotide arm versus control arm,P = 0.03. Wilcoxon test for VOD-associated multiple organ failure anddeath in patients with VOD at Day + 30: defibrotide arm versus controlarm, P = 0.2.

TABLE 5 Patients with Drug-Related Adverse Events According to TreatmentArm bySystem Organ Class and Preferred Term.* Defibrotide Arm ControlArm† (N = 177) (N = 176) System Organ Class/Abnormality No. (%) No. (%)Blood and lymphatic system disorders Coagulopathy 0 (0) 1 (1)Gastrointestinal disorders Gastrointestinal hemorrhage 2 (1) 3 (2)Abdominal pain 1 (1) 0 (0) Diarrhea hemorrhagic 1 (1) 0 (0) Hematemesis1 (1) 0 (0) Mouth hemorrhage 1 (1) 0 (0) Nausea 1 (1) 0 (0) Uppergastrointestinal hemorrhage 0 (0) 1 (1) Vomiting 1 (1) 0 (0)Investigations Activated partial thromboplastin time 0 (0) 2 (1)prolonged Prothrombin time prolonged 1 (1) 1 (1) Respiratory, thoracic,and mediastinal disorders Epistaxis 2 (1) 1 (1) Hemothorax 1 (1) 1 (1)Pulmonary hemorrhage 0 (0) 1 (1) Vascular disorders Hemorrhage 1 (1) 1(1) Microangiopathy 0 (0) 1 (1) *Related adverse events are thoseclassified by the investigator as possibly, likely or certainly relatedto study drug. †Adverse events reported following a diagnosis ofveno-occlusive disease (VOD); defibrotide received as treatment for VOD.

1. Defibrotide for use in prophylaxis and/or treatment of Graft versusHost Disease (GVHD) in humans.
 2. Defibrotide according to claim 1,characterised in that said Graft versus Host Disease is acute orchronic, preferably acute.
 3. Defibrotide according to claim 1,characterised in that it is administered before and/or afterhematopoietic stem cell transplantation (HSCT).
 4. Defibrotide accordingto claim 3, characterised in that said hematopoietic stem celltransplantation (HSCT) is allogenic hematopoietic stem celltransplantation.
 5. Defibrotide according to claim 1, characterised inthat it is administered in a dose ranging from 10 to 60 mg/kg per day,preferably from 20 to 40 mg/kg per day.
 6. Defibrotide according toclaim 5, characterised in that it is administered in a dose of about 25mg/kg per day.
 7. Defibrotide according to claim 1, characterised inthat it is administered in a single or repeated dose per day, preferablyin four (4) doses per day.
 8. Defibrotide according to claim 1,characterised in that is administered intravenously.
 9. Defibrotideaccording to claim 1, characterised in that is administered in aqueousform.
 10. Defibrotide according to claim 1, characterised in that it isobtained by extraction from animal and/or vegetable tissues, preferablyfrom mammalian organs.